Fig 1: Expression of HE4, ANXA2 and P-gp in ovarian cancer cells (CAOV3, SKOV3 and OVCAR3). (A) Western blot analysis of HE4, ANXA2 and P-gp expression in three cell lines. (B) Comparison of HE4, ANXA2 and P-gp protein levels in three cell lines. (C) Immunocytochemistry detection of P-gp expression in three cell lines. Magnification, ×400. *P<0.05. HE4; human epididymis protein 4; P-gp, P-glycoprotein
Fig 2: Inhibitory regulation of hsa-miR-129-5p by HE4 via interactions with ANXA2. (A) Expression of five miRNAs expression before and after transfection, detected by reverse transcription-quantitative polymerase chain reaction assays. (B) Expression of hsa-miR-129-5p in cells before and after treatment by different antibodies or active protein at different time points. The cells of each group were treated as same as described for Fig. 3H. *P<0.05. HE4; human epididymis protein 4; microRNA/miR, microRNAs.
Fig 3: Interaction of HE4 with ANXA2 enhances P-gp expression and promotes resistance to adriamycin in CAOV3 cells. (A) Expression of HE4, ANXA2 and P-gp before and after transfection, detected by western blotting. (B) Comparison of HE4, ANXA2 and P-gp protein levels before and after transfection. (C) Expression of HE4, ANXA2 and P-gp before and after transfection, detected by RT-qPCR assays. (D) Cells were treated with 0–10 µg/ml adriamycin for 24 h before and after transfection, at which time the cells were subjected to MTT assay to measure viability. Results are displayed as percent survival of vehicle-treated cells. Error bars represent the standard deviation of biological replicates. (E) Flow cytometry detection of the expression of P-gp on the cell membrane before and after transfection. (F) Measurement of adriamycin accumulation by flow cytometry before and after transfection, cells of different groups were pretreated with complete medium containing or not containing verapamil (50 µM) for 1 h. After pretreatment, cells were incubated with adriamycin in culture medium. (G) Measurement of MFI of adriamycin by flow cytometry in cells before and after transfection. (H) Expression of P-gp in cells before and after treatment by different antibodies or active protein at different time points, detected by western blotting. Group 1, CAOV3 cells; group 2, CAOV3 cells treated with HE4 active protein; group 3, CAOV3 cells treated with ANXA2 active protein; group 4, CAOV3 cells treated with HE4 antibody; group 5, CAOV3 cells treated with ANXA2 antibody; group 6, CAOV3 cells treated with HE4 active protein and ANXA2 antibody; group 7, CAOV3-A2-L cells; group 8, CAOV3-A2-L cells treated with HE4 active protein; and group 9, CAOV3 cells treated with IgG. (I) Comparison of P-gp protein levels before and after treatment by different antibodies or active protein at different time points. The cells of each group were treated as same as (H). (J) Expression of P-gp in cells before and after treatment by different antibodies or active protein at different time points, detected by RT-qPCR assays. The cells of each group were treated as same as (H). *P<0.05. HE4; human epididymis protein 4; P-gp, P-glycoprotein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MFI, mean fluorescence intensity; VER, verapamil.
Fig 4: Human epididymis protein 4, ANXA2 and P-glycoprotein genes were commonly enriched in the signaling pathway involved in the regulation of the actin cytoskeleton.
Fig 5: Interaction of HE4, ANXA2 and P-gp in CAOV3 cells. (A) Protein lysates of CAOV3 cells were precipitated using HE4, ANXA2 and P-gp-specific antibodies. WB revealed co-expression of HE4 with ANXA2, and ANXA2 with P-gp, respectively. (B) Double-labeling immunofluorescence showed the colocalization of HE4 or ANXA2 with P-gp in CAOV3 cells. Magnification, ×400. HE4; human epididymis protein 4; P-gp, P-glycoprotein; WB, western blotting; TCL, total cell lysate; NTC, negative control, the primary antibody was replaced by rabbit IgG; IP, immunoprecipitate.
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